Germline stat depletion (A, D–G; Tj Gal4/UAS HA-stat; stat^F/stat^85c9) or global stat depletion (B; stat^F/stat^85c9) eight days after stat temperature inactivation. Hub (asterisk and Fas3, white); germ cells (vasa, magenta); DNA (blue). (A) Testes with germline stat depletion maintained CySCs (Zfh-1, green), and germ cells. (B) Testes with global stat depletion exhibited germ cell loss (lack of magenta cells) and CySC loss (Zfh-1, green; note, a few CySCs always persisted near the hub). (C) Global stat depletion led to progressive germ cell loss, first the more undifferentiated spermatogonia (blue; this category includes GSCs/single germ cells) followed by spermatocytes (orange), while most testes with germline stat depletion maintained spermatogonia (blue) indefinitely. Similar results were obtained with three CySC lineage Gal4 drivers and three strong stat alleles with the stat^ts allele (). (D–G) Stat-depleted germ cells continually progressed through differentiation, shown by normal expression of the spermatogonial differentiation marker Bam (D, green), the appearance of branching fusomes through germ cell cysts (E, α-spectrin, green), synchronously cycling germ cell cysts (F, Edu pulse-labeled cells, green), and expression of the spermatocyte marker Ptb (G, green). Bar:10μm.

Germ cells (vasa, magenta), Hub (asterisk or Fas3, white). (A) Wild-type GSCs are tightly associated with the hub. (B) Two days after germline stat depletion (Tj Gal4/UAS HA-stat; stat^F/stat^85c9), GSCs became displaced from the hub. (C–F) 8 days after germline stat depletion, germ cells nearest the hub accumulate M5-4 lacZ (C, white, arrows), have spherical or dumbbell-shaped spectrosomes (D, α-spectrin, green, arrows; D′ shows α-spectrin alone), and undergo mitosis as individual cells (E, phosphohistoneH3, green, arrow; a mitotic CySC is also present), all consistent with GSC identity. M5-4 lacZ (C, white) also accumulates in the hub (arrowheads), but not in CySCs (Tj, green), indicating CySCs do not take on hub fate. (F) stat-depleted GSCs fail to reliably orient mitotic spindles away from the hub (centrosomin reveals centrosomes, green). Two mitotic germ cells outlined, one oriented (arrow) and one non-oriented (arrowhead). (G) With germline stat depletion, the hub became surrounded by CySCs (Zfh-1, green). (H) Testes with Gal4-driven stat in the CySC lineage (Tj Gal4/UAS stat; stat^85c9/+) undergo modest GSC loss, but still maintain GSCs at the hub (arrows, Zfh-1, green). (I) Germline RNAi knockdown of stat (nos gal4 VP16 VDRC 106980) caused GSCs to be displaced from the hub, and the hub became surrounded by CySCs (Zfh-1, green). Bar:10μm.

(A) Most control GSCs (stat^F/stat^85c9, 18° permissive temperature) have one centrosome oriented toward the hub interface (arrowheads, γ-tubulin (green) shows centrosomes; vasa (magenta) shows germ cells). (B) Upon stat depletion (sixteen hours after upshift) many GSC centrosomes were misoriented (arrows), while others were not (arrowheads). These GSCs are Bam-negative (A′ and B′, white shows Bam with E-cadherin marking the hub). (C) In control GSCs (Nos Gal4 VP16/UAS E-cadherinGFP; stat^85c9/+, 16hrs 29°), E-cadherinGFP (green) localizes to the hub interface (vasa, magenta; fas3, white). (D) Upon sixteen hour stat depletion (Nos Gal4 VP16/UAS E-cadherinGFP; stat^F/stat^85c9) E-cadherinGFP was lost (arrows) or highly reduced (arrowheads) from the hub interface. E-cadherinGFP only (C′, D′). (E) Germline Par3GFP (green) colocalizes with E-cadherin (magenta) at hub-GSC interface in controls (Nos Gal4 VP16/UAS Par3GFP; stat^85c9/+, 16hrs 29°). (F) Upon sixteen hour stat depletion (Nos Gal4 VP16/UAS Par3GFP; stat^F/stat^85c9) Par3GFP (green) delocalized, with only modest interface enrichment remaining. Quantified in . (G) Germline UAS E-cadherin expression in stat^ts testes delayed germ cell loss compared to stat^ts alone or with UAS Par3 expression. Asterisk denotes hub. Bar:10μm.

Germ cells (vasa, magenta), hub (asterisk). Eight days after germline stat depletion, the germ cells near the hub accumulated phospho-Smad (A, green, arrows; A′ pSmad alone) and Dad-lacZ (B, green, arrows; Zfh-1 in white; B′ Dad-lacZ alone). (C) Sustained zfh-1 expression in the cyst lineage (eyaA3 Gal4/UAS zfh-1; tubGal80^ts; twelve days) generated excess GSCs (magenta) and these accumulated Dad-lacZ. DNA (blue). (D) Most testes with germline stat depletion retain spermatogonia (blue), but dampening the BMP pathway by coexpression of UAS sog^CR1 (P<0.05 for 8d) or kekkon5 (P<0.0001 for 8d) reduced the proportion of testes with rescued germ cells. (E) Model for self-renewal in the testis niche. Bar:10μm.